Identification of Potential Lmx1b‐Regulated Enhancers During Limb Development

Lmx1b is a LIM homeodomain transcription factor required for developmental patterning. In the limb, itsexpression is restricted to the dorsal limb mesenchyme and is responsible for limb dorsalization. Mice lacking Lmx1b function develop ventral‐ventral limbs, whereas ectopic ventral expression of Lmx1b in chick leads to the development of dorsal‐dorsal limbs. In humans, LMX1B mutation is associated with Nail‐Patella Syndrome and is characterized by nail dysplasia, absent or hypoplastic patellae, and progressive renal disease. Despite the striking effect of Lmx1b on limb dorsalization, no direct limb targets have been described. In order to identify direct limb targets, Lmx1b‐targeted chromatin imunoprecipitation followed by massive parallel sequencing (ChIP‐seq) was performed during murine limb development (E12.5). Sites of Lmx1b binding were further analyzed for conservation across divergent vertebrate species to enhance discovery of Lmx1b‐associated regulatory regions. To determine genes directly regulated by Lmx1b, we compared ChIP‐seq data with genes differentially expressed in the presence of Lmx1b. We identified 590 genomic sites of Lmx1b binding and defined its binding motif. Conservation amongst human, mice, and chicken was evident in 169 sites. Of these conserved noncoding regions (CNR), 34 were associated with 37 genes differentially expressed by Lmx1b using microarray analysis at E12.5. Thus, we identified 34 potential enhancer elements and 3 have shown to drive activity in the developing limb. Interestingly, none of the 590 Lmx1b binding sites were found in gene promoters, indicating that Lmx1b acts at distant regulatory regions, likely modulating the expression of target genes rather than controlling their induction.